Anti-CD5 (H65) h(CD28-CD3ζ) CAR, pCDCAR1 (CAR-LC173)

CAR Construction : H65-CD28-CD3ζ

Fig.1 Cytotoxicity assay in vitro.

Cytotoxicity of CD19 CAR- and CD5 CAR-transduced T cells against T-ALL and T-lymphoma cell lines was assessed in a 5-hour Cr release assay.

Mamonkin, M., Rouce, R. H., Tashiro, H., & Brenner, M. K. (2015). A T-cell–directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood, The Journal of the American Society of Hematology, 126(8), 983-992.

CAR Construction : H65-CD28-CD3ζ

Fig.2 Determination of cytokines release.

Production of IFN-γ and TNF-α by CD4+(top) and CD8+(bottom) T cells transduced with CD19 CAR or CD5 CAR was measured by intracellular cytokine staining.

Mamonkin, M., Rouce, R. H., Tashiro, H., & Brenner, M. K. (2015). A T-cell–directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood, The Journal of the American Society of Hematology, 126(8), 983-992.

CAR Construction : H65-CD28-CD3ζ

Fig.3 CD5 CAR T cells control progression of T-ALL in xenograft mouse models.

Jurkat-FFluc cells (Fig.A-E) or CCRF-CEM-FFluc cells (Fig.F-H) were IV injected followed by IV injection of CAR T cells on days 3 and 6 postimplantation (Fig.A, F) or on day 6 and 9 post-implantation (Fig.C).

Mamonkin, M., Rouce, R. H., Tashiro, H., & Brenner, M. K. (2015). A T-cell–directed chimeric antigen receptor for the selective treatment of T-cell malignancies. Blood, The Journal of the American Society of Hematology, 126(8), 983-992.

CAR Construction : H65-41BB-CD3ζ

Fig.4 The immunophenotype of CD5CAR T cells.

The majority of CD5KO anti-CD5 CAR-T cells displayed a naive-like surface phenotype that might have an enhanced capacity for expansion, differentiation, and self-renewal upon antigen stimulation.

Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., ... & Zhou, J. (2021). The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Molecular Therapy, 29(9), 2707-2722.

CAR Construction : H65-41BB-CD3ζ

Fig.5 Cytotoxicity assay in vitro.

Anti-CD5 CAR-T cells exhibited cytotoxicity in vitro when co-incubated with malignant T cell lines with moderate expression of CD5 antigen at relatively low effector to target (E:T) ratios

Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., ... & Zhou, J. (2021). The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Molecular Therapy, 29(9), 2707-2722.

CAR Construction : H65-41BB-CD3ζ

Fig.6 Cytokine secretion and repeated stimulation assay of CD5-targeting CAR-T cells in vitro.

CAR-T cells produced the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon (IFN)-γ and elevated levels of interleukin-2 (IL-2) release when co-incubated with CCRF-CEM cells.

Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., ... & Zhou, J. (2021). The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Molecular Therapy, 29(9), 2707-2722.

CAR Construction : H65-41BB-CD3ζ

Fig.7 The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model established by CCRF-CEM.

A mouse tumor model of T cell acute lymphoblastic leukemia (T-ALL) by tail intravenous injection of CCRF-CEM-ffLuc cells was established to verify the in vivo efficacy of CD5 CAR-T cells.

Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., ... & Zhou, J. (2021). The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Molecular Therapy, 29(9), 2707-2722.

CAR Construction : H65-41BB-CD3ζ

Fig.8 The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model established by SUP-T1.

A xenograft mouse tumor model was established by SUP-T1 cells with moderate CD5 expression to verify the in vivo efficacy of CD5 CAR-T cells.

Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., ... & Zhou, J. (2021). The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Molecular Therapy, 29(9), 2707-2722.

CAR Construction :

Fig.9 The binding affinity measurement of H65 antibody to CD5 antigen.

The affinity of H65 to recombinant human CD5 was determined using bio-layer interferometry.

Dai, Z., Mu, W., Zhao, Y., Jia, X., Liu, J., Wei, Q., ... & Zhou, J. (2021). The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains. Molecular Therapy, 29(9), 2707-2722.